Cloning of a novel G-protein coupled 7TM Receptor

ABSTRACT

HNHCI32 polypeptides and polynucleotides and methods for producing such polypeptides by recombinant techniques are disclosed. Also disclosed are methods for utilizing HNHCI32 polypeptides and polynucleotides in the design of protocols for the treatment of infections such as bacterial, fungal, protozoan and viral infections, particularly infections caused by HIV-1 or HIV-2; pain; cancers; anorexia; bulimia; asthma; Parkinson&#39;s disease; acute heart failure; hypotension; hypertension; urinary retention; osteoporosis; angina pectoris; myocardial infarction; ulcers; asthma; allergies; benign prostatic hypertrophy; and psychotic and neurological disorders, including anxiety, schizophrenia, manic depression, delirium, dementia, severe mental retardation and dyskinesias, such as Huntington&#39;s disease or Gilles dela Tourett&#39;s syndrome, among others and diagnostic assays for such conditions.

FIELD OF INVENTION

[0001] This invention relates to newly identified polynucleotides,polypeptides encoded by them and to the use of such polynucleotides andpolypeptides, and to their production. More particularly, thepolynucleotides and polypeptides of the present invention relate toG-Protein coupled receptor, hereinafter referred to as HNHCI32. Theinvention also relates to inhibiting or activating the action of suchpolynucleotides and polypeptides.

BACKGROUND OF THE INVENTION

[0002] It is well established that many medically significant biologicalprocesses are mediated by proteins participating in signal transductionpathways that involve G-proteins and/or second messengers, e.g., cAMP(Lefkowitz, Nature, 1991, 351:353-354). Herein these proteins arereferred to as proteins participating in pathways with G-proteins or PPGproteins. Some examples of these proteins include the GPC receptors,such as those for adrenergic agents and dopamine (Kobilka, B. K., etal., Proc. Natl Acad. Sci., USA, 1987, 84:46-50; Kobilka, B. K., et al.,Science, 1987, 238:650-656; Bunzow, J. R., et al., Nature, 1988,336:783-787), G-proteins themselves, effector proteins, e.g.,phospholipase C, adenyl cyclase, and phosphodiesterase, and actuatorproteins, e.g., protein kinase A and protein kinase C (Simon, M. I., etal., Science, 1991, 252:802-8).

[0003] For example, in one form of signal transduction, the effect ofhormone binding is activation of the enzyme, adenylate cyclase, insidethe cell. Enzyme activation by hormones is dependent on the presence ofthe nucleotide GTP. GTP also influences hormone binding. A G-proteinconnects the hormone receptor to adenylate cyclase. G-protein was shownto exchange GTP for bound GDP when activated by a hormone receptor. TheGTP-carrying form then binds to activated adenylate cyclase. Hydrolysisof GTP to GDP, catalyzed by the G-protein itself, returns the G-proteinto its basal, inactive form. Thus, the G-protein serves a dual role, asan intermediate that relays the signal from receptor to effector, and asa clock that controls the duration of the signal.

[0004] The membrane protein gene superfamily of G-protein coupledreceptors has been characterized as having seven putative transmembranedomains. The domains are believed to represent transmembrane a-helicesconnected by extracellular or cytoplasmic loops. G-protein coupledreceptors include a wide range of biologically active receptors, such ashormone, viral, growth factor and neuroreceptors.

[0005] G-protein coupled receptors (otherwise known as 7TM receptors)have been characterized as including these seven conserved hydrophobicstretches of about 20 to 30 amino acids, connecting at least eightdivergent hydrophilic loops. The G-protein family of coupled receptorsincludes dopamine receptors which bind to neuroleptic drugs used fortreating psychotic and neurological disorders. Other examples of membersof this family include, but are not limited to, calcitonin, adrenergic,endothelin, cAMP, adenosine, muscarinic, acetylcholine, serotonin,histamine, thrombin, kinin, follicle stimulating hormone, opsins,endothelial differentiation gene-1, rhodopsins, odorant, andcytomegalovirus receptors.

[0006] Most G-protein coupled receptors have single conserved cysteineresidues in each of the first two extracellular loops which formdisulfide bonds that are believed to stabilize functional proteinstructure. The 7 transmembrane regions are designated as TM 1, TM2, TM3,TM4, TM5, TM6, and TM7. TM3 has been implicated in signal transduction.

[0007] Phosphorylation and lipidation (palmitylation or farnesylation)of cysteine residues can influence signal transduction of some G-proteincoupled receptors. Most G-protein coupled receptors contain potentialphosphorylation sites within the third cytoplasmic loop and/or thecarboxy terminus. For several G-protein coupled receptors, such as theb-adrenoreceptor, phosphorylation by protein kinase A and/or specificreceptor kinases mediates receptor desensitization.

[0008] For some receptors, the ligand binding sites of G-protein coupledreceptors are believed to comprise hydrophilic sockets formed by severalG-protein coupled receptor transmembrane domains, said socket beingsurrounded by hydrophobic residues of the G-protein coupled receptors.The hydrophilic side of each G-protein coupled receptor transmembranehelix is postulated to face inward and form polar ligand binding site.TM3 has been implicated in several G-protein coupled receptors as havinga ligand binding site, such as the TM3 aspartate residue. TM5 serines, aTM6 asparagine and TM6 or TM7 phenylalanines or tyrosines are alsoimplicated in ligand binding.

[0009] G-protein coupled receptors can be intracellularly coupled byheterotrimeric G-proteins to various intracellular enzymes, ion channelsand transporters (see, Johnson et al., Endoc. Rev., 1989, 10:317-331)Different G-protein a-subunits preferentially stimulate particulareffectors to modulate various biological functions in a cell.Phosphorylation of cytoplasmic residues of G-protein coupled receptorshave been identified as an important mechanism for the regulation ofG-protein coupling of some G-protein coupled receptors. G-proteincoupled receptors are found in numerous sites within a mammalian host.

[0010] Over the past 15 years, nearly 350 therapeutic agents targeting 7transmembrane (7 TM) receptors have been successfully introduced ontothe market.

[0011] This indicates that these receptors have an established, provenhistory as therapeutic targets. Clearly there is a need foridentification and characterization of further receptors which can playa role in preventing, ameliorating or correcting dysfunctions ordiseases, including, but not limited to, infections such as bacterial,fungal, protozoan and viral infections, particularly infections causedby HIV-1 or HIV-2; pain; cancers; anorexia; bulimia; asthma; Parkinson'sdisease; acute heart failure; hypotension; hypertension; urinaryretention; osteoporosis; angina pectoris; myocardial infarction; ulcers;asthma; allergies; benign prostatic hypertrophy; and psychotic andneurological disorders, including anxiety, schizophrenia, manicdepression, delirium, dementia, severe mental retardation anddyskinesias, such as Huntington's disease or Gilles dela Tourett'ssyndrome.

SUMMARY OF THE INVENTION

[0012] In one aspect, the invention relates to HNHCI32 polypeptides andrecombinant materials and methods for their production. Another aspectof the invention relates to methods for using such HNHCI32 polypeptidesand polynucleotides. Such uses include the treatment of infections suchas bacterial, fungal, protozoan and viral infections, particularlyinfections caused by HIV-1 or HIV-2; pain; cancers; anorexia; bulimia;asthma; Parkinson's disease; acute heart failure; hypotension;hypertension; urinary retention; osteoporosis; angina pectoris;myocardial infarction; ulcers; asthma; allergies; benign prostatichypertrophy; and psychotic and neurological disorders, includinganxiety, schizophrenia, manic depression, delirium, dementia, severemental retardation and dyskinesias, such as Huntington's disease orGilles dela Tourett's syndrome, among others. In still another aspect,the invention relates to methods to identify agonists and antagonistsusing the materials provided by the invention, and treating conditionsassociated with HNHCI32 imbalance with the identified compounds. Yetanother aspect of the invention relates to diagnostic assays fordetecting diseases associated with inappropriate HNHCI32 activity orlevels.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013] FIG. I shows the nucleotide and deduced amino acid sequence ofhuman HNHCI32. SEQ ID NOS: 1 and 2.

DESCRIPTION OF THE INVENTION

[0014] Definitions

[0015] The following definitions are provided to facilitateunderstanding of certain terms used frequently herein.

[0016] “HNHCI32” refers generally to a polypeptide having the amino acidsequence set forth in SEQ ID NO:2, or an allelic variant thereof.

[0017] “Receptor Activity” or “Biological Activity of the Receptor”refers to the metabolic or physiologic function of said HNHCI32including similar activities or improved activities or these activitieswith decreased undesirable side-effects. Also included are antigenic andimmunogenic activities of said HNHCI32.

[0018] “HNHCI32 polypeptides” refers to polypeptides with amino acidsequences sufficiently similar to HNHCI32 sequences, preferablyexhibiting at least one biological activity of the receptor.

[0019] “HNHCI32 gene” refers to a polynucleotide having the nucleotidesequence set forth in SEQ ID NO: 1 or allelic variants thereof and/ortheir complements.

[0020] “HNHCI32 polynucleotides” refers to polynucleotides containing anucleotide sequence which encodes a HNHCI32 polypeptide or fragmentthereof, or a nucleotide sequence which has at least 80% identity to anucleotide sequence encoding the polypeptide of SEQ ID NO:2 or thecorresponding fragment thereof, or a nucleotide sequence which hassufficient identity to a nucleotide sequence contained in SEQ ID NO:1 tohybridize under conditions useable for amplification or for use as aprobe or marker.

[0021] “Antibodies” as used herein includes polyclonal and monoclonalantibodies, chimeric, single chain, and humanized antibodies, as well asFab fragments, including the products of an Fab or other immunoglobulinexpression library.

[0022] “Isolated” means altered “by the hand of man” from the naturalstate. If an “isolated” composition or substance occurs in nature, ithas been changed or removed from its original environment, or both. Forexample, a polynucleotide or a polypeptide naturally present in a livinganimal is not “isolated,” but the same polynucleotide or polypeptideseparated from the coexisting materials of its natural state is“isolated”, as the term is employed herein.

[0023] “Polynucleotide” generally refers to any polyribonucleotide orpolydeoxribonucleotide, which may be unmodified RNA or DNA or modifiedRNA or DNA. “Polynucleotides” include, without limitation single- anddouble-stranded DNA, DNA that is a mixture of single- anddouble-stranded regions, single- and double-stranded RNA, and RNA thatis mixture of single- and double-stranded regions, hybrid moleculescomprising DNA and RNA that may be single-stranded or, more typically,double-stranded or a mixture of single- and double-stranded regions. Inaddition, “polynucleotide” refers to triple-stranded regions comprisingRNA or DNA or both RNA and DNA. The term polynucleotide also includesDNAs or RNAs containing one or more modified bases and DNAs or RNAs withbackbones modified for stability or for other reasons. “Modified” basesinclude, for example, tritylated bases and unusual bases such asinosine. A variety of modifications has been made to DNA and RNA; thus,“polynucleotide” embraces chemically, enzymatically or metabolicallymodified forms of polynucleotides as typically found in nature, as wellas the chemical forms of DNA and RNA characteristic of viruses andcells. “Polynucleotide” also embraces relatively short polynucleotides,often referred to as oligonucleotides.

[0024] “Polypeptide” refers to any peptide or protein comprising two ormore amino acids joined to each other by peptide bonds or modifiedpeptide bonds, i.e., peptide isosteres. “Polypeptide” refers to bothshort chains, commonly referred to as peptides, oligopeptides oroligomers, and to longer chains, generally referred to as proteins.Polypeptides may contain amino acids other than the 20 gene-encodedamino acids. “Polypeptides” include amino acid sequences modified eitherby natural processes, such as posttranslational processing, or bychemical modification techniques which are well known in the art. Suchmodifications are well described in basic texts and in more detailedmonographs, as well as in a voluminous research literature.Modifications can occur anywhere in a polypeptide, including the peptidebackbone, the amino acid side-chains and the amino or carboxyl termini.It will be appreciated that the same type of modification may be presentin the same or varying degrees at several sites in a given polypeptide.Also, a given polypeptide may contain many types of modifications.Polypeptides may be branched as a result of ubiquitination, and they maybe cyclic, with or without branching. Cyclic, branched and branchedcyclic polypeptides may result from posttranslation natural processes ormay be made by synthetic methods. Modifications include acetylation,acylation, ADP-ribosylation, amidation, covalent attachment of flavin,covalent attachment of a heme moiety, covalent attachment of anucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, covalent attachment of phosphotidylinositol,cross-linking, cyclization, disulfide bond formation, demethylation,formation of covalent cross-links, formation of cystine, formation ofpyroglutamate, formylation, gamma-carboxylation, glycosylation, GPIanchor formation, hydroxylation, iodination, methylation,myristoylation, oxidation, proteolytic processing, phosphorylation,prenylation, racemization, selenoylation, sulfation, transfer-RNAmediated addition of amino acids to proteins such as arginylation, andubiquitination. See, for instance, PROTEINS-STRUCTURE AND MOLECULARPROPERTIES, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, NewYork, 1993 and Wold, F., Posttranslational Protein Modifications:Perspectives and Prospects, pgs. 1-12 in POSTTRANSLATIONAL COVALENTMODIFICATION OF PROTEINS, B. C. Johnson, Ed., Academic Press, New York,1983; Seifter et al, “Analysis for protein modifications and nonproteincofactors”, Meth Enzymol (1990) 182:626-646 and Rattan et al., “ProteinSynthesis: Posttranslational Modifications and Aging”, Ann NY Acad Sci(1992) 663:48-62.

[0025] “Variant” as the term is used herein, is a polynucleotide orpolypeptide that differs from a reference polynucleotide or polypeptiderespectively, but retains essential properties. A typical variant of apolynucleotide differs in nucleotide sequence from another, referencepolynucleotide. Changes in the nucleotide sequence of the variant may ormay not alter the amino acid sequence of a polypeptide encoded by thereference polynucleotide. Nucleotide changes may result in amino acidsubstitutions, additions, deletions, fusions and truncations in thepolypeptide encoded by the reference sequence, as discussed below. Atypical variant of a polypeptide differs in amino acid sequence fromanother, reference polypeptide. Generally, differences are limited sothat the sequences of the reference polypeptide and the variant areclosely similar overall and, in many regions, identical. A variant andreference polypeptide may differ in amino acid sequence by one or moresubstitutions, additions, deletions in any combination. A substituted orinserted amino acid residue may or may not be one encoded by the geneticcode. A variant of a polynucleotide or polypeptide may be a naturallyoccurring such as an allelic variant, or it may be a variant that is notknown to occur naturally. Non-naturally occurring variants ofpolynucleotides and polypeptides may be made by mutagenesis techniquesor by direct synthesis.

[0026] “Identity” is a measure of the identity of nucleotide sequencesor amino acid sequences. In general, the sequences are aligned so thatthe highest order match is obtained. “Identity” per se has anart-recognized meaning and can be calculated using published techniques.See, e.g.: (COMPUTATIONAL MOLECULAR BIOLOGY, Lesk, A. M., ed., OxfordUniversity Press, New York, 1988; BIOCOMPUTING: INFORMATICS AND GENOMEPROJECTS, Smith, D. W., ed., Academic Press, New York, 1993; COMPUTERANALYSIS OF SEQUENCE DATA, PART I, Griffin, A. M., and Griffin, H. G.,eds., Humana Press, New Jersey, 1994; SEQUENCE ANALYSIS IN MOLECULARBIOLOGY, von Heinje, G., Academic Press, 1987; and SEQUENCE ANALYSISPRIMER, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York,1991). While there exist a number of methods to measure identity betweentwo polynucleotide or polypeptide sequences, the term “identity” is wellknown to skilled artisans (Carillo, H., and Lipton, D., SIAM J AppliedMath (1988) 48:1073). Methods commonly employed to determine identity orsimilarity between two sequences include, but are not limited to, thosedisclosed in Guide to Huge Computers, Martin J. Bishop, ed., AcademicPress, San Diego, 1994, and Carillo, H., and Lipton, D., SIAM J AppliedMath (1988) 48:1073. Methods to determine identity and similarity arecodified in computer programs. Preferred computer program methods todetermine identity and similarity between two sequences include, but arenot limited to, GCS program package (Devereux, J., et al., Nucleic AcidsResearch (1984) 12(1):387), BLASTP, BLASTN, FASTA (Atschul, S. F. etal., J Molec Biol (1990) 215:403).

[0027] Polypeptides of the Invention

[0028] The HNHCI32 polypeptides of the present invention include thepolypeptide of SEQ ID NO:2 (in particular the mature polypeptide) aswell as HNHCI32 polypeptides and which have at least 80% identity to thepolypeptide of SEQ ID NO:2 or the relevant portion and more preferablyat least 90% identity, and still more preferably at least 95% identity,and even still more preferably at least 99% identity to SEQ ID NO:2.

[0029] The HNHCI32 polypeptides may be in the form of the “mature”protein or may be a part of a larger protein such as a fusion protein.It is often advantageous to include an additional amino acid sequencewhich contains secretory or leader sequences, pro-sequences, sequenceswhich aid in purification such as multiple histidine residues, or anadditional sequence for stability during recombinant production.

[0030] Biologically active fragments of the HNHCI32 polypeptides arealso included in the invention. A fragment is a polypeptide having anamino acid sequence that entirely is the same as part, but not all, ofthe amino acid sequence of the aforementioned HNHCI32 polypeptides. Aswith HNHCI32 polypeptides, fragments may be “free-standing,” orcomprised within a larger polypeptide of which they form a part orregion, most preferably as a single continuous region. Representativeexamples of polypeptide fragments of the invention, include, forexample, fragments from about amino acid number 1-20, 21-40, 41-60,61-80, 81-100, and 101 to the end of HNHCI32 polypeptide. In thiscontext “about” includes the particularly recited ranges larger orsmaller by several, 5, 4, 3, 2 or 1 amino acid at either extreme or atboth extremes.

[0031] Preferred fragments include, for example, truncation polypeptideshaving the amino acid sequence of HNHCI32 polypeptides, except fordeletion of a continuous series of residues that includes the aminoterminus, or a continuous series of residues that includes the carboxylterminus or deletion of two continuous series of residues, one includingthe amino terminus and one including the carboxyl terminus. Alsopreferred are fragments characterized by structural or functionalattributes such as fragments that comprise alpha-helix and alpha-helixforming regions, beta-sheet and beta-sheet-forming regions, turn andturn-forming regions, coil and coil-forming regions, hydrophilicregions, hydrophobic regions, alpha amphipathic regions, betaamphipathic regions, flexible regions, surface-forming regions,substrate binding region, and high antigenic index regions. Biologicallyactive fragments are those that mediate receptor activity, includingthose with a similar activity or an improved activity, or with adecreased undesirable activity. Also included are those that areantigenic or immunogenic in an animal, especially in a human.

[0032] Thus, the polypeptides of the invention include polypeptideshaving an amino acid sequence at least 80% identical to that of SEQ IDNO:2 or fragments thereof with at least 80% identity to thecorresponding fragment of SEQ ID NO:2. Preferably, all of thesepolypeptides retain the biological activity of the receptor, includingantigenic activity. Included in this group are variants of the definedsequence and fragments. Preferred variants are those that vary from thereferents by conservative amino acid substitutions—i.e., those thatsubstitute a residue with another of like characteristics. Typical suchsubstitutions are among Ala, Val, Leu and Ile; among Ser and Thr; amongthe acidic residues Asp and Glu; among Asn and Gln; and among the basicresidues Lys and Arg; or aromatic residues Phe and Tyr. Particularlypreferred are variants in which several, 5-10, 1-5, or 1-2 amino acidsare substituted, deleted, or added in any combination.

[0033] The HNHCI32 polypeptides of the invention can be prepared in anysuitable manner. Such polypeptides include isolated naturally occurringpolypeptides, recombinantly produced polypeptides, syntheticallyproduced polypeptides, or polypeptides produced by a combination ofthese methods. Means for preparing such polypeptides are well understoodin the art.

[0034] Polynucleotides of the Invention

[0035] Another aspect of the invention relates to isolatedpolynucleotides which encode the HNHCI32 polypeptides andpolynucleotides closely related thereto.

[0036] HNHCI32 of the invention is structurally related to otherproteins of the G-Protein coupled receptor, as shown by the results ofsequencing the cDNA encoding human HNHCI32. The cDNA sequence containsan open reading frame encoding a protein of 343 with a deduced molecularweight of 39.07 kDa. HNHCI32 of FIG. 1 (SEQ ID NO:2) has about 27.2%identity (using Fasta) in 184 amino acid residues with human 5HT1Dreceptor (M. W. Hamblin and M. A Metcalf, Mol. Pharmcol. 40: 14314 1481991). Furthermore HNHCI32 (Seq. ID NO:2) is 26.2% identical to Ratalpha-1B adrenergic receptor over 210 amino acid residues (M. M. Voigt,et al., Nucleic Acid Res. 18: 1053-1055 1990). HNHCI32 gene of FIG. 1(SEQ ID NO:1) has about 54% identity (using blast) in 288 nucleotideresidues with human Seretonin receptor 5HT1E (J. M. Zgobick, et al.,Mol. Pharmacol. 42: 180-185, 1992). Furthermore, HNHCI32 of SEQ ID NO:1has about 54% identity in 158 nucleotide residues with human S31 genefor Seretonin receptor ( F. O. Levy, et al., FEBS lett. 296(2): 201-2061992).

[0037] One polynucleotide of the present invention encoding HNHCI32 maybe obtained using standard cloning and screening, from a cDNA libraryderived from mRNA in cells of human Neutrophil Induced, using theexpressed sequence tag (EST) analysis (Adams, M. D., et al. Science(1991) 252:1651-1656; Adams, M. D. et al., Nature, (1992) 355:632-634;Adams, M. D., et al., Nature (1995) 377 Supp:3-174). Polynucleotides ofthe invention can also be obtained from natural sources such as genomicDNA libraries or can be synthesized using well known and commerciallyavailable techniques.

[0038] Thus, the nucleotide sequence encoding HNHCI32 polypeptides maybe identical over its entire length to the coding sequence in FIG. 1(SEQ ID NO:1), or may be a degenerate form of this nucleotide sequenceencoding the polypeptide of SEQ ID NO:2, or may be highly identical to anucleotide sequence that encodes the polypeptide of SEQ ID NO:2.Preferably, the polynucleotides of the invention contain a nucleotidesequence that is highly identical, at least 80% identical, with anucleotide sequence encoding a HNHCI32 polypeptide, or at least 80%identical with the encoding nucleotide sequence set forth in FIG. 1 (SEQID NO:1), or at least 80% identical to a nucleotide sequence encodingthe polypeptide of SEQ ID NO:2.

[0039] When the polynucleotides of the invention are used for therecombinant production of HNHCI32 polypeptide, the polynucleotide mayinclude the coding sequence for the mature polypeptide or a fragmentthereof, by itself; the coding sequence for the mature polypeptide orfragment in reading frame with other coding sequences, such as thoseencoding a leader or secretory sequence, a pre-, or pro- or prepro-protein sequence, or other fusion peptide portions. For example, amarker sequence which facilitates purification of the fused polypeptidecan be encoded. In certain preferred embodiments of this aspect of theinvention, the marker sequence is a hexa-histidine peptide, as providedin the pQE vector (Qiagen, Inc.) and described in Gentz et al, Proc NatlAcad Sci USA (1989) 86:821-824, or is an HA tag. The polynucleotide mayalso contain non-coding 5′ and 3′ sequences, such as transcribed,non-translated sequences, splicing and polyadenylation signals, ribosomebinding sites and sequences that stabilize mRNA.

[0040] Among particularly preferred embodiments of the invention arepolynucleotides encoding HNHCI32 polypeptides having the amino acidsequence of set out in FIG. 1 (SEQ ID NO:2) and variants thereof.

[0041] Further preferred embodiments are polynucleotides encodingHNHCI32 variants that have the amino acid sequence of the HNHCI32 ofFIG. 1 (SEQ ID NO:2) in which several, 5-10, 1-5, 1-3, 1-2 or 1 aminoacid residues are substituted, deleted or added, in any combination.

[0042] Further preferred embodiments of the invention arepolynucleotides that are at least 80% identical over their entire lengthto a polynucleotide encoding the HNHCI32 polypeptide having the aminoacid sequence set out in FIG. 1 (SEQ ID NO:2), and polynucleotides whichare complementary to such polynucleotides. In this regard,polynucleotides at least 80% identical over their entire length to thesame are particularly preferred, and those with at least 90% areespecially preferred. Furthermore, those with at least 97% are highlypreferred and those with at least 98-99% are most highly preferred, withat least 99% being the most preferred.

[0043] The present invention further relates to polynucleotides thathybridize to the herein above-described sequences. In this regard, thepresent invention especially relates to polynucleotides which hybridizeunder stringent conditions to the herein above-describedpolynucleotides. As herein used, the term “stringent conditions” meanshybridization will occur only if there is at least 95% and preferably atleast 97% identity between the sequences.

[0044] Polynucleotides of the invention, which are sufficientlyidentical to a nucleotide sequence contained in SEQ ID NO:1, may be usedas hybridization probes for cDNA and genomnic DNA, to isolatefull-length cDNAs and genomic clones encoding HNHCI32 and to isolatecDNA and genomic clones of other genes that have a high sequencesimilarity to the HNHCI32 gene. Such hybridization techniques are knownto those of skill in the art. Typically these nucleotide sequences are70% identical, preferably 80% identical, more preferably 90% identicalto that of the referent. The probes generally will comprise at least 15nucleotides. Preferably, such probes will have at least 30 nucleotidesand may have at least 50 nucleotides. Particularly preferred probes willrange between 30 and 50 nucleotides.

[0045] The polynucleotides and polypeptides of the present invention maybe employed as research reagents and materials for discovery oftreatments and diagnostics to animal and human disease.

[0046] Vectors, Host Cells, Expression

[0047] The present invention also relates to vectors which comprise apolynucleotide or polynucleotides of the present invention, and hostcells which are genetically engineered with vectors of the invention andto the production of polypeptides of the invention by recombinanttechniques. Cell-free translation systems can also be employed toproduce such proteins using RNAs derived from the DNA constructs of thepresent invention.

[0048] For recombinant production, host cells can be geneticallyengineered to incorporate expression systems or portions thereof forpolynucleotides of the present invention. Introduction ofpolynucleotides into host cells can be effected by methods described inmany standard laboratory manuals, such as Davis et al, BASIC METHODS INMOLECULAR BIOLOGY (1986) and Sambrook et al, MOLECULAR CLONING: ALABORATORYMANUAL, 2nd Ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (1989) such as calcium phosphate transfection,DEAE-dextran mediated transfection, transvection, microinjection,cationic lipid-mediated transfection, electroporation, transduction,scrape loading, ballistic introduction or infection.

[0049] Representative examples of appropriate hosts include bacterialcells, such as streptococci, staphylococci E coli, Streptomyces andBacillus subtilis cells; fungal cells, such as yeast cells andAspergillus cells; insect cells such as Drosophila S2 and Spodoptera Sf9cells; animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, 293 andBowes melanoma cells; and plant cells.

[0050] A great variety of expression systems can be used. Such systemsinclude, among others, chromosomal, episomal and virus-derived systems,e.g., vectors derived from bacterial plasmids, from bacteriophage, fromtransposons, from yeast episomes, from insertion elements, from yeastchromosomal elements, from viruses such as baculoviruses, papovaviruses, such as SV40, vaccinia viruses. adenoviruses, fowl pox viruses,pseudorabies viruses and retroviruses, and vectors derived fromcombinations thereof, such as those derived from plasmid andbacteriophage genetic elements, such as cosmids and phagemids. Theexpression systems may contain control regions that regulate as well asengender expression. Generally, any system or vector suitable tomaintain, propagate or express polynucleotides to produce a polypeptidein a host may be used. The appropriate nucleotide sequence may beinserted into an expression system by any of a variety of well-known androutine techniques, such as, for example, those set forth in Sambrook etal., MOLECULAR CLONING, A LABORATORYMANUAL (supra).

[0051] For secretion of the translated protein into the lumen of theendoplasmic reticulum, into the periplasmic space or into theextracellular environment, appropriate secretion signals may beincorporated into the desired polypeptide. These signals may beendogenous to the polypeptide or they may be heterologous signals.

[0052] If the HNHCI32 polypeptide is to be expressed for use inscreening assays, generally, it is preferred that the polypeptide beproduced at the surface of the cell. In this event, the cells may beharvested prior to use in the screening assay. If HNHCI32 polypeptide issecreted into the medium, the medium can be recovered in order torecover and purify the polypeptide; if produced intracellularly, thecells must first be lysed before the polypeptide is recovered.

[0053] HNHCI32 polypeptides can be recovered and purified fromrecombinant cell cultures by well-known methods including ammoniumsulfate or ethanol precipitation, acid extraction, anion or cationexchange chromatography, phosphocellulose chromatography, hydrophobicinteraction chromatography, affinity chromatography, hydroxylapatitechromatography and lectin chromatography. Most preferably, highperformance liquid chromatography is employed for purification. Wellknown techniques for refolding proteins may be employed to regenerateactive conformation when the polypeptide is denatured during isolationand or purification.

[0054] Diagnostic Assays

[0055] This invention also relates to the use of HNHCI32 polynucleotidesfor use as diagnostic reagents. Detection of a mutated form of HNHCI32gene associated with a dysfunction will provide a diagnostic tool thatcan add to or define a diagnosis of a disease or susceptibility to adisease which results from under-expression, over-expression or alteredexpression of HNHCI32. Individuals carrying mutations in the HNHCI32gene may be detected at the DNA level by a variety of techniques.

[0056] Nucleic acids for diagnosis may be obtained from a subject'scells, such as from blood, urine, saliva, tissue biopsy or autopsymaterial. The genomic DNA may be used directly for detection or may beamplified enzymatically by using PCR or other amplification techniquesprior to analysis. RNA or cDNA may also be used in similar fashion.Deletions and insertions can be detected by a change in size of theamplified product in comparison to the normal genotype. Point mutationscan be identified by hybridizing amplified DNA to labeled HNHCI32nucleotide sequences. Perfectly matched sequences can be distinguishedfrom mismatched duplexes by RNase digestion or by differences in meltingtemperatures. DNA sequence differences may also be detected byalterations in electrophoretic mobility of DNA fragments in gels, withor without denaturing agents, or by direct DNA sequencing. See, e.g.,Myers et al, Science (1985) 230:1242. Sequence changes at specificlocations may also be revealed by nuclease protection assays, such asRNase and S1 protection or the chemical cleavage method. See Cotton etal, Proc Natl Acad Sci USA (1985) 85: 4397-4401.

[0057] The diagnostic assays offer a process for diagnosing ordetermining a susceptibility to infections such as bacterial, fungal,protozoan and viral infections, particularly infections caused by HIV-1or HIV-2; pain; cancers; anorexia; bulimia; asthma; Parkinson's disease;acute heart failure; hypotension; hypertension; urinary retention;osteoporosis; angina pectoris; myocardial infarction; ulcers; asthma;allergies; benign prostatic hypertrophy; and psychotic and neurologicaldisorders, including anxiety, schizophrenia, manic depression, delirium,dementia, severe mental retardation and dyskinesias, such asHuntington's disease or Gilles dela Tourett's syndrome through detectionof mutation in the HNHCI32 gene by the methods described.

[0058] In addition, infections such as bacterial, fungal, protozoan andviral infections, particularly infections caused by HIV-1 or HIV-2;pain; cancers; anorexia; bulimia; asthma; Parkinson's disease; acuteheart failure; hypotension; hypertension; urinary retention;osteoporosis; angina pectoris; myocardial infarction; ulcers; asthma;allergies; benign prostatic hypertrophy; and psychotic and neurologicaldisorders, including anxiety, schizophrenia, manic depression, delirium,dementia, severe mental retardation and dyskinesias, such asHuntington's disease or Gilles dela Tourett's syndrome, can be diagnosedby methods comprising determining from a sample derived from a subjectan abnormally decreased or increased level of HNHCI32 polypeptide orHNHCI32 mRNA. Decreased or increased expression can be measured at theRNA level using any of the methods well known in the art for thequantitation of polynucleotides, such as, for example, PCR RT-PCR, RNaseprotection, Northern blotting and other hybridization methods. Assaytechniques that can be used to determine levels of a protein, such as anHNHCI32, in a sample derived from a host are well-known to those ofskill in the art. Such assay methods include radioimmunoassays,competitive-binding assays, Western Blot analysis and ELISA assays.

[0059] Chromosome Assays

[0060] The nucleotice sequences of the present invention are alsovaluable for chromosome identification. The sequence is specificallytargeted to and can hybridize with a particular location on anindividual human chromosome. The mapping of relevant sequences tochromosomes according to the present invention is an important firststep in correlating those sequences with gene associated disease. Once asequence has been mapped to a precise chromosomal location, the physicalposition of the sequence on the chromosome can be correlated withgenetic map data. Such data are found, for example, in V. McKusick,Mendelian Inheritance in Man (available on line through Johns HopkinsUniversity Welch Medical Library). The relationship between genes anddiseases that have been mapped to the same chromosomal region are thenidentified through linkage analysis (coinheritance of physicallyadjacent genes). The differences in the cDNA or genomic sequence betweenaffected and unaffected individuals can also be determined. If amutation is observed in some or all of the affected individuals but notin any normal individuals, then the mutation is likely to be thecausative agent of the disease.

[0061] Antibodies

[0062] The polypeptides of the invention or their fragments or analogsthereof, or cells expressing them can also be used as immunogens toproduce antibodies immunospecific for the HNHCI32 polypeptides. The term“immunospecific” means that the antibodies have substantially greateraffinity for the polypeptides of the invention than their affinity forother related polypeptides in the prior art.

[0063] Antibodies generated against the HNHCI32 polypeptides can beobtained by administering the polypeptides or epitope-bearing fragments,analogs or cells to an animal, preferably a nonhuman, using routineprotocols. For preparation of monoclonal antibodies, any technique whichprovides antibodies produced by continuous cell line cultures can beused. Examples include the hybridoma technique (Kohler, G. and Milstein,C., Nature (1975) 256:495-497), the trioma technique, the human B-cellhybridorna technique (Kozbor et al., Immnunology Today (1983) 4:72) andthe EBV-hybridoma technique (Cole et al, MONOCLONAL ANTIBODIES ANDCANCER THERAPY, pp.77-96, Alan R. Liss, Inc., 1985).

[0064] Techniques for the production of single chain antibodies (U.S.Pat. No. 4,946,778) can also be adapted to produce single chainantibodies to polypeptides of this invention. Also, transgenic mice, orother organisms including other mammals, may be used to expresshumanized antibodies.

[0065] The above-described antibodies may be employed to isolate or toidentify clones expressing the polypeptide or to purify the polypeptidesby affinity chromatography.

[0066] Antibodies against HNHCI32 polypeptides may also be employed totreat infections such as bacterial, fungal, protozoan and viralinfections, particularly infections caused by HIV-1 or HIV-2; pain;cancers; anorexia; bulimia; asthma; Parkinson's disease; acute heartfailure; hypotension; hypertension; urinary retention; osteoporosis;angina pectoris; myocardial infarction; ulcers; asthma; allergies;benign prostatic hypertrophy; and psychotic and neurological disorders,including anxiety, schizophrenia, manic depression, delirium, dementia,severe mental retardation and dyskinesias, such as Huntington's diseaseor Gilles dela Tourett's syndrome, among others.

[0067] Vaccines

[0068] Another aspect of the invention relates to a method for inducingan immunological response in a mammal which comprises inoculating themammal with HNHCI32 polypeptide, or a fragment thereof adequate toproduce antibody and/or T cell immune response to protect said animalfrom infections such as bacterial, fungal, protozoan and viralinfections, particularly infections caused by HIV-1 or HIV-2; pain;cancers; anorexia; bulimia; asthma; Parkinson's disease; acute heartfailure; hypotension; hypertension; urinary retention; osteoporosis;angina pectoris; myocardial infarction; ulcers; asthma; allergies;benign prostatic hypertrophy; and psychotic and neurological disorders,including anxiety, schizophrenia, manic depression, delirium, dementia,severe mental retardation and dyskinesias, such as Huntington's diseaseor Gilles dela Tourett's syndrome, among others. Yet another aspect ofthe invention relates to a method of inducing immunological response ina mammal which comprises, delivering HNHCI32 gene via a vector directingexpression of HNHCI32 polypeptide in vivo in order to induce such animmunological response to produce antibody to protect said animal fromdiseases.

[0069] Further aspect of the invention relates to animmunological/vaccine formulation (composition) which when introducedinto a mammalian host, induces an immunological response in that mammalto a HNHCI32 polypeptide wherein the composition comprises a HNHCI32polypeptide or HNHCI32 gene. The vaccine formulation may furthercomprise a suitable carrier. Since HNHCI32 polypeptide may be brokendown in the stomach, it is preferably administered parenterally(including subcutaneous, intramuscular, intravenous, intradermal etc.injection). Formulations suitable for parenteral administration includeaqueous and non-aqueous sterile injection solutions which may containanti-oxidants, buffers, bacteriostats and solutes which render theformulation instonic with the blood of the recipient; and aqueous andnor-aqueous sterile suspensions which may include suspending agents orthickening agents. The formulations may be presented in unit-dose ormulti-dose containers, for example, sealed ampoules and vials and may bestored in a freeze-dried condition requiring only the addition of thesterile liquid carrier immediately prior to use. The vaccine formulationmay also include adjuvant systems for enhancing the immunogenicity ofthe formulation, such as oil-in water systems and other systems known inthe art. The dosage will depend on the specific activity of the vaccineand can be readily determined by routine experimentation.

[0070] Screening Assays

[0071] The HNHCI32 of the present invention may be employed in ascreening process for compounds which bind the receptor and whichactivate (agonists) or inhibit activation of (antagonists) the receptorpolypeptide of the present invention. Thus, polypeptides of theinvention may also be used to assess the binding of small moleculesubstrates and ligands in, for example, cells, cell-free preparations,chemical libraries, and natural product mixtures. These substrates andligands may be natural substrates and ligands or may be structural orfunctional mimetics. See Coligan et al, Current Protocols in Immunology1(2):Chapter 5 (1991).

[0072] HNHCI32 proteins are ubiquitous in the mammalian host and areresponsible for many biological functions, including many pathologies.Accordingly, it is desirous to find compounds and drugs which stimulateHNHCI32 on the one hand and which can inhibit the function of HNHCI32 onthe other hand. In general, agonists are employed for therapeutic andprophylactic purposes for such conditions as infections such asbacterial, fungal, protozoan and viral infections, particularlyinfections caused by HIV-1 or HIV-2; pain; cancers; anorexia; bulimia;asthma; Parkinson's disease; acute heart failure; hypotension;hypertension; urinary retention; osteoporosis; angina pectoris;myocardial infarction; ulcers; asthma; allergies; benign prostatichypertrophy; and psychotic and neurological disorders, includinganxiety, schizophrenia, manic depression, delirium, dementia, severemental retardation and dyskinesias, such as Huntington's disease orGilles dela Tourett's syndrome. Antagonists may be employed for avariety of therapeutic and prophylactic purposes for such conditions asinfections such as bacterial, fungal, protozoan and viral infections,particularly infections caused by HIV-1 or HIV-2; pain; cancers;anorexia; bulimia; asthma; Parkinson's disease; acute heart failure;hypotension; hypertension; urinary retention; osteoporosis; anginapectoris; myocardial infarction; ulcers; asthma; allergies; benignprostatic hypertrophy; and psychotic and neurological disorders,including anxiety, schizophrenia, manic depression, delirium, dementia,severe mental retardation and dyskinesias, such as Huntington's diseaseor Gilles dela Tourett's syndrome.

[0073] In general, such screening procedures involve producingappropriate cells which express the receptor polypeptide of the presentinvention on the surface thereof. Such cells include cells from mammals,yeast. Drosophila or E. coli. Cells expressing the receptor (or cellmembrane containing the expressed receptor) are then contacted with atest compound to observe binding, or stimulation or inhibition of afunctional response.

[0074] One screening technique includes the use of cells which expressreceptor of this invention (for example, transfected CHO cells) in asystem which measures extracellular pH or intracellular calcium changescaused by receptor activation. In this technique, compounds may becontacted with cells expressing the receptor polypeptide of the presentinvention. A second messenger response, e.g., signal transduction, pHchanges, or changes in calcium level, is then measured to determinewhether the potential compound activates or inhibits the receptor.

[0075] Another method involves screening for receptor inhibitors bydetermining inhibition or stimulation of receptor-mediated cAMP and/oradenylate cyclase accumulation. Such a method involves transfecting aeukaryotic cell with the receptor of this invention to express thereceptor on the cell surface. The cell is then exposed to potentialantagonists in the presence of the receptor of this invention. Theamount of cAMP accumulation is then measured. If the potentialantagonist binds the receptor, and thus inhibits receptor binding, thelevels of receptor-mediated cAMP, or adenylate cyclase, activity will bereduced or increased.

[0076] Another methods for detecting agonists or antagonists for thereceptor of the present invention is the yeast based technology asdescribed in U.S. Pat. No. 5,482,835.

[0077] The assays may simply test binding of a candidate compoundwherein adherence to the cells bearing the receptor is detected by meansof a label directly or indirectly associated with the candidate compoundor in an assay involving competition with a labeled competitor. Further,these assays may test whether the candidate compound results in a signalgenerated by activation of the receptor, using detection systemsappropriate to the cells bearing the receptor at their surfaces.Inhibitors of activation are generally assayed in the presence of aknown agonist and the effect on activation by the agonist by thepresence of the candidate compound is observed. Standard methods forconducting such screening assays are well understood in the art.

[0078] Examples of potential HNHCI32 antagonists include antibodies or,in some cases, oligonucleotides or proteins which are closely related tothe ligand of the HNHCI32, e.g., a fragment of the ligand, or smallmolecules which bind to the receptor but do not elicit a response, sothat the activity of the receptor is prevented.

[0079] Prophylactic and Therapeutic Methods

[0080] This invention provides methods of treating an abnormalconditions related to both an excess of and insufficient amounts ofHNHCI32 activity.

[0081] If the activity of HNHCI32 is in excess, several approaches areavailable. One approach comprises administering to a subject aninhibitor compound (antagonist) as hereinabove described along with apharmaceutically acceptable carrier in an amount effective to inhibitactivation by blocking binding of ligands to the HNHCI32, or byinhibiting a second signal, and thereby alleviating the abnormalcondition.

[0082] In another approach, soluble forms of HNHCI32 polypeptides stillcapable of binding the ligand in competition with endogenous HNHCI32 maybe administered. Typical embodiments of such competitors comprisefragments of the HNHCI32 polypeptide.

[0083] In still another approach, expression of the gene encodingendogenous HNHCI32 can be inhibited using expression blockingtechniques. Known such techniques involve the use of antisensesequences, either internally generated or separately administered. See,for example, O' Connor, J Neurochem (1991) 56:560 inOligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRCPress, Boca Raton, Fla. (1988). Alternatively, oligonucleotides whichform triple helices with the gene can be supplied. See, for example, Leeet al, Nucleic Acids Res (1979) 6:3073; Cooney et al., Science (1988)241:456; Dervan et al, Science (1991) 251:1360. These oligomers can beadministered per se or the relevant oligomers can be expressed in vivo.

[0084] For treating abnormal conditions related to an under-expressionof HNHCI32 and its activity, several approaches are also available. Oneapproach comprises administering to a subject a therapeuticallyeffective amount of a compound which activates HNHCI32, i.e., an agonistas described above, in combination with a pharmaceutically acceptablecarrier, to thereby alleviate the abnormal condition. Alternatively,gene therapy may be employed to effect the endogenous production ofHNHCI32 by the relevant cells in the subject. For example, apolynucleotide of the invention may be engineered for expression in areplication defective retroviral vector, as discussed above. Theretroviral expression construct may then be isolated and introduced intoa packaging cell transduced with a retroviral plasmid vector containingRNA encoding a polypeptide of the present invention such that thepackaging cell now produces infectious viral particles containing thegene of interest. These producer cells may be administered to a subjectfor engineering cells in vivo and expression of the polypeptide in vivo.For overview of gene therapy, see Chapter 20, Gene Therapy and otherMolecular Genetic-based Therapeutic Approaches, (and references citedtherein) in Human Molecular Genetics, T Strachan and A P Read, BIOSScientific Publishers Ltd (1996).

[0085] Formulation and Administration

[0086] Peptides, such as the soluble form of HNHCI32 polypeptides, andagonists and antagonist peptides or small molecules, may be formulatedin combination with a suitable pharmaceutical carrier. Such formulationscomprise a therapeutically effective amount of the polypeptide orcompound, and a pharmaceutically acceptable carrier or excipient. Suchcarriers include but are not limited to, saline, buffered saline,dextrose, water, glycerol, ethanol, and combinations thereof.Formulation should suit the mode of administration, and is well withinthe skill of the art. The invention further relates to pharmaceuticalpacks and kits comprising one or more containers filled with one or moreof the ingredients of the aforementioned compositions of the invention.

[0087] Polypeptides and other compounds of the present invention may beemployed alone or in conjunction with other compounds, such astherapeutic compounds.

[0088] Preferred forms of systemic administration of the pharmaceuticalcompositions include injection, typically by intravenous injection.Other injection routes, such as subcutaneous, intramuscular, orintraperitoneal, can be used. Alternative means for systemicadministration include transmucosal and transdermal administration usingpenetrants such as bile salts or fusidic acids or other detergents. Inaddition, if properly formulated in enteric or encapsulatedformulations, oral administration may also be possible. Administrationof these compounds may also be topical and/or localized, in the form ofsalves, pastes, gels and the like.

[0089] The dosage range required depends on the choice of peptide, theroute of administration, the nature of the formulation, the nature ofthe subject's condition, and the judgment of the attending practitioner.Suitable dosages, however, are in the range of 0.1-100 μg/kg of subject.Wide variations in the needed dosage, however, are to be expected inview of the variety of compounds available and the differingefficiencies of various routes of administration. For example, oraladministration would be expected to require higher dosages thanadministration by intravenous injection. Variations in these dosagelevels can be adjusted using standard empirical routines foroptimization, as is well understood in the art.

[0090] Polypeptides used in treatment can also be generated endogenouslyin the subject, in treatment modalities often referred to as “genetherapy” as described above. Thus, for example, cells from a subject maybe engineered with a polynucleotide, such as a DNA or RNA, to encode apolypeptide ex vivo, and for example, by the use of a retroviral plasmidvector. The cells are then introduced into the subject.

EXAMPLES

[0091] The examples below are carried out using standard techniques,which are well known and routine to those of skill in the art, exceptwhere otherwise described in detail. The examples illustrate, but do notlimit the invention.

Example 1

[0092] A partial clone (ATG 781, HGS EST # 691136) was initiallyidentified through random searches of the Human Genome Sciences database. This partial clone (594 bp) showed significant homology ( 31.4%over 121 aa) to Alpha-2 Adrenergic receptor. To get the full lengthcDNA: A total of 1M plaques were screened from a Human Genomic Placentalibrary (Stratagene, LaJolla Calif., Cat. # 946206) using a 450 bp PCRfragment corresponding to above partial clone as a probe. The genomiclibrary screening procedure is described by (Elgin, et al. Strategies 4:8-9, 1991). The probes were α-32P labeled, using Random Primed LabelingKit (Boheringer Manheim, Germany, Cat. # 1585584 ) and purified byrunning over Sephadex G-50 columns (Pharmacia Biotech. Cat. #17-0855-02) The hybirdization and washing conditions were according to(J. Sambrook, E. F. Fritch and T. Maniatis (1989) A Laboratory ManaulSecond. Ed. Vol. 1 pp. 2.69-2.81 Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y.). 2 Positives phage clones obtained form abovescreen were further purified. Southern analysis were carried out ondigested phage DNA and an 8.0 kb Sac I fragment hybirdized for bothclones. This fragment was subcloned into pBlueScript KS vector and thesequence of the insert was determined by automated sequencer. A total of2273 bp were sequenced, this includes an open reading frame enconding apeptide of 343 aa. Fasta analysis show this peptide to have highhomology to Amenergic receptor (5HT1D). Furthermore hydrophobicity plotof this peptide using the Lasergene Protean program showed the seven TMspaning hydrophobic domains, typical of all 7TM receptors.

[0093] To conform the coding region of this clone, a Human BrainHippocampus Marathon Ready cDNA( Clontech, Palo Alto, Calif.) was usedas a template to amplify an 800bp fragment corrosponding to the 5′region of HNHCI32, using reverse gene specific oligos and forward cDNAspecific Marathon ready cDNA oligos. This fragment was directly gelpurified and sequenced. The sequence was identical to the genomic cloneobtained above.

Example 2: Mammalian Cell Expression

[0094] The receptors of the present invention are expressed in eitherhuman embryonic kidney 293 (HEK293) cells or adherent dhfr CHO cells. Tomaximize receptor expression, typically all 5′ and 3′ untranslatedregions (UTRs) are removed from the receptor cDNA prior to insertioninto a pCDN or pCDNA3 vector. The cells are transfected with individualreceptor cDNAs by lipofectin and selected in the presence of 400 mg/mlG418. After 3 weeks of selection, individual clones are picked andexpanded for further analysis. HEK293 or CHO cells transfected with thevector alone serve as negative controls. To isolate cell lines stablyexpressing the individual receptors, about 24 clones are typicallyselected and analyzed by Northern blot analysis. Receptor mRNAs aregenerally detectably in about 50% of the G418-resistant clones analyzed.

Example 3 Ligand bank for binding and functional assays.

[0095] A bank of over 200 putative receptor ligands has been assembledfor screening. The bank comprises: transmitters, hormones andchernokines known to act via a human seven transmembrane (7TM) receptor;naturally occurring compounds which may be putative agonists for a human7TM receptor, non-mammalian, biologically active peptides for which amammalian counterpart has not yet been identified; and compounds notfound in nature, but which activate 7TM receptors with unknown naturalligands. This bank is used to initially screen the receptor for knownligands, using both functional (i.e. calcium, cAMP, microphysiometer,oocyte electrophysiology, etc, see below) as well as binding assays.

Example 4: Ligand Binding Assays

[0096] Ligand binding assays provide a direct method for ascertainingreceptor pharmacology and are adaptable to a high throughput format. Thepurified ligand for a receptor is radiolabeled to high specific activity(50-2000 Ci/mmol) for binding studies. A determination is then made thatthe process of radiolabeling does not diminish the activity of theligand towards its receptor. Assay conditions for buffers, ions, pH andother modulators such as nucleotides are optimized to establish aworkable signal to noise ratio for both membrane and whole cell receptorsources. For these assays, specific receptor binding is defined as totalassociated radioactivity minus the radioactivity measured in thepresence of an excess of unlabeled competing ligand. Where possible,more than one competing ligand is used to define residual nonspecificbinding.

Example 5: Functional Assay in Xenopus Oocytes

[0097] Capped RNA transcripts from linearized plasmid templates encodingthe receptor cDNAs of the invention are synthesized in vitro with RNApolyrnerases in accordance with standard procedures. In vitrotranscripts are suspended in water at a final concentration of 0.2mg/ml. Ovarian lobes are removed from adult female toads, Stage Vdefolliculated oocytes are obtained, and RNA transcripts (10 ng/oocyte)are injected in a 50 nl bolus using a microinjection apparatus. Twoelectrode voltage clamps are used to measure the currents fromindividual Xenopus oocytes in response to agonist exposure.. Recordingsare made in Ca2+ free Barth's medium at room temperature. The Xenopussystem can be used to screen known ligands and tissue/cell extracts foractivating ligands.

Example 6: Microphysiometric Assays

[0098] Activation of a wide variety of secondary messenger systemsresults in extrusion of small amounts of acid from a cell. The acidformed is largely as a result of the increased metabolic activityrequired to fuel the intracellular signaling process. The pH changes inthe media surrounding the cell are very small but are detectable by theCYTOSENSOR microphysiometer (Molecular Devices Ltd., Menlo Park,Calif.). The CYTOSENSOR is thus capable of detecting the activation of areceptor which is coupled to an energy utilizing intracellular signalingpathway such as the G-protein coupled receptor of the present invention.

Example 7: Extract/Cell Supernatant Screening

[0099] A large number of mammalian receptors exist for which thereremains, as yet, no cognate activating ligand (agonist). Thus, activeligands for these receptors may not be included within the ligands banksas identified to date. Accordingly, the 7TM receptor of the invention isalso functionally screened (using calcium, cAMP, microphysiometer,oocyte electrophysiology, etc., functional screens) against tissueextracts to identify natural ligands. Extracts that produce positivefunctional responses can be sequencially subfractionated until anactivating ligand is isolated identified.

Example 8: Calcium and cAMP Functional Assays

[0100] 7TM receptors which are expressed in HEK 293 cells have beenshown to be coupled functionally to activation of PLC and calciummobilization and/or cAMP stimuation or inhibition. Basal calcium levelsin the HEK 293 cells in receptor-transfected or vector control cellswere observed to be in the normal, 100 nM to 200 nM, range. HEK 293cells expressing recombinant receptors are loaded with fura 2 and in asingle day >150 selected ligands or tissue/cell extracts are evaluatedfor agonist induced calcium mobilization. Similarly, HEK 293 cellsexpressing recombinant receptors are evaluated for the stimulation orinhibition of cAMP production using standard cAMP quantitation assays.Agonists presenting a calcium transient or cAMP flucuation are tested invector control cells to determine if the response is unique to thetransfected cells expressing receptor.

1 2 1 2273 DNA HOMO SAPIENS 1 ggagtggctt ttccagattg atgtgtattccggaagcaaa tagcaaaggc aagcgtgacc 60 atttattgga agtttttatt tatgccatttgtccaccctc ctttattcag cccactgtga 120 ccaaaaggag tcatgtgaga tttgagtaactgggtctgtg agtgtggaat ttgtaatttt 180 tattgtctct cagggatgga acatatgacatggcttcact agttagaatc tcagagaagc 240 tataagactt aagaaataag ggaaggagcatgaagagtag aggacaggag aggcagaggg 300 2 343 PRT HOMO SAPIENS 2 Met AspLeu Thr Tyr Ile Pro Glu Asp Leu Ser Ser Cys Pro Lys Phe 1 5 10 15 ValAsn Lys Ile Leu Ser Ser His Gln Pro Leu Phe Ser Cys Pro Gly 20 25 30 AspAsn Val Phe Gly Tyr Asp Trp Ser His Asp Tyr Pro Leu Phe Gly 35 40 45 AsnLeu Val Ile Met Val Ser Ile Ser His Phe Lys Gln Leu His Ser 50 55 60 ProThr Asn Phe Leu Ile Leu Ser Met Ala Thr Thr Asp Phe Leu Leu 65 70 75 80Gly Phe Val Ile Met Pro Tyr Ser Ile Met Arg Ser Val Glu Ser Cys 85 90 95Trp Tyr Phe Gly Asp Gly Phe Cys Lys Phe His Thr Ser Phe Asp Met 100 105110 Met Leu Arg Leu Thr Ser Ile Phe His Leu Cys Ser Ile Ala Ile Asp 115120 125 Arg Phe Tyr Ala Val Cys Tyr Pro Leu His Tyr Thr Thr Lys Met Thr130 135 140 Asn Ser Thr Ile Lys Gln Leu Leu Ala Phe Cys Trp Ser Val ProAla 145 150 155 160 Leu Phe Ser Phe Gly Leu Val Leu Ser Glu Ala Asp ValSer Gly Met 165 170 175 Gln Ser Tyr Lys Ile Leu Val Ala Cys Phe Asn PheCys Ala Leu Thr 180 185 190 Phe Asn Lys Phe Trp Gly Thr Ile Leu Phe ThrThr Cys Phe Phe Thr 195 200 205 Pro Gly Ser Ile Met Val Gly Ile Tyr GlyLys Ile Phe Ile Val Ser 210 215 220 Lys Gln His Ala Arg Val Ile Ser HisVal Pro Glu Asn Thr Lys Gly 225 230 235 240 Ala Val Lys Lys His Leu SerLys Lys Lys Asp Arg Lys Ala Ala Lys 245 250 255 Thr Leu Gly Ile Val MetGly Val Phe Leu Ala Cys Trp Leu Pro Cys 260 265 270 Phe Leu Ala Val LeuIle Asp Pro Tyr Leu Asp Tyr Ser Thr Pro Ile 275 280 285 Leu Ile Leu AspLeu Leu Val Trp Leu Arg Tyr Phe Asn Ser Thr Cys 290 295 300 Asn Pro LeuIle His Gly Phe Phe Asn Pro Trp Phe Gln Lys Ala Phe 305 310 315 320 LysTyr Ile Val Ser Gly Lys Ile Phe Ser Ser His Ser Glu Thr Ala 325 330 335Asn Leu Phe Pro Glu Ala His 340

What is claimed is:
 1. An isolated polynucleotide comprising anucleotide sequence that has at least 80% identity to a nucleotidesequence encoding the polypetide of SEQ ID NO:2 or the correspondingfragment thereof; or a nucleotide sequence complementary to saidnucleotide sequence.
 2. The polynucleotide of claim 1 which is DNA orRNA.
 3. The polynucleotide of claim 1 wherein said nucleotide sequenceis at least 80% identical to that contained in SEQ ID NO:1.
 4. Thepolynucleotide of claim 3 wherein said nucleotide sequence is containedin SEQ ID NO:1.
 5. The polynucleotide of claim 1 wherein said encodingnucleotide sequence encodes the polypeptide of SEQ ID NO:2 or a fragmentthereof.
 6. A polynucleotide probe or primer comprising at least 15contiguous nucleotides of the polynucleotide of claim 3 .
 7. A DNA orRNA molecule comprising an expression system wherein said expressionsystem is capable of producing a HNHCI32 or a fragment thereof having atleast 80% identity with a nucleotide sequence encoding the polypeptideof SEQ ID NO:2 or said fragment when said expression system is presentin a compatible host cell.
 8. A host cell comprising the expressionsystem of claim 7 .
 9. A process for producing a HNHCI32 polypeptide orfragment comprising culturing a host of claim 8 and under conditionssufficient for the production of said polypeptide or fragment.
 10. Theprocess of claim 9 wherein said polypeptide or fragment is expressed atthe surface of said cell.
 11. Cells produced by the process of claim
 10. 12. The process of claim 9 which further includes recovering thepolypeptide or fragment from the culture.
 13. A process for producing acell which produces a HNHCI32 polypeptide or a fragment thereofcomprising transforming or transfecting a host cell with the expressionsystem of claim 7 such that the host cell, under appropriate cultureconditions, produces a HNHCI32 polypeptide or fragment.
 14. A HNHCI32polypeptide or a fragment thereof comprising an amino acid sequencewhich is at least 80% identical to the amino acid sequence contained inSEQ ID NO:2.
 15. The polypeptide of claim 14 which comprises the aminoacid sequence of SEQ ID NO:2, or a fragment thereof.
 16. A HNHCI32polypeptide or fragment prepared by the method of claim 12 .
 17. Anantibody immunospecific for the HNHCI32 polypeptide of claim 14 .
 18. Amethod for the treatment of a subject in need of enhanced HNHCI32activity comprising: (a) administering to the subject a therapeuticallyeffective amount of an agonist to said receptor; and/or (b) providing tothe subject HNHCI32 polynucleotide in a form so as to effect productionof said receptor activity in vivo.
 19. A method for the treatment of asubject having need to inhibit HNHCI32 activity comprising: (a)administering to the subject a therapeutically effective amount of anantagonist to said receptor; and/or (b) administering to the subject anucleic acid molecule that inhibits the expression of the nucleotidesequence encoding said receptor; and/or (c) administering to the subjecta therapeutically effective amount of a polypeptide that competes withsaid receptor for its ligand.
 20. A process for diagnosing a disease ora susceptibility to a disease in a subject related to expression oractivity of HNHCI32 in a subject comprising: (a) determining thepresence or absence of a mutation in the nucleotide sequence encodingsaid HNHCI32 in the genome of said subject; and/or (b) analyzing for thepresence or amount of the HNHCI32 expression in a sample derived fromsaid subject.
 21. A method for identifying compounds which bind toHNHCI32 comprising: (a) contacting cells of claim 11 with a candidatecompound; and (b) assessing the ability of said candidate compound tobind to said cells.
 22. The method of claim 21 which further includesdetermining whether the candidate compound effects a signal generated byactivation of the HNHCI32 polypeptide at the surface of the cell,wherein a candidate compound which effects production of said signal isidentified as an agonist.
 23. An agonist identified by the method ofclaim 22 .
 24. The method of claim 21 which further includes contactingsaid cell with a known agonist for said HNHCI32; and determining whetherthe signal generated by said agonist is diminished in the presence ofsaid candidate compound, wherein a candidate compound which effects adiminution in said signal is identified as an antagonist for saidHNHCI32.
 25. An antagonist identified by the method of claim 24 .